The Role of the Cytoskeleton in Cell Body Enlargement, Increased

BMC Neuroscience BioMed Central Research article Open Access The role of the cytoskeleton in cell body enlargement, increased nuclear eccentricity and chromatolysis in axotomized spinal motor neurons David L McIlwain*1 and Victoria B Hoke2 Address: 1Department of Cell and Molecular Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA and 2Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707, USA Email: David L McIlwain* - [email protected]; Victoria B Hoke - [email protected] * Corresponding author Published: 17 March 2005 Received: 10 November 2004 Accepted: 17 March 2005 BMC Neuroscience 2005, 6:19 doi:10.1186/1471-2202-6-19 This article is available from: http://www.biomedcentral.com/1471-2202/6/19 © 2005 McIlwain and Hoke; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: When spinal motor axons are injured, the nucleolus, nucleus and cell body of the injured cell transiently increase in size, the nucleus becomes more eccentrically placed, and the organization of polyribosomes into Nissl bodies is temporarily disrupted. The mechanisms for these classical morphological responses to axotomy have not been satisfactorily explained. Results: In this study we address the role of the cell body cytoskeleton in these structural changes. We show that the cytoskeleton of uninjured lumbar motor neuron cell bodies maintains nucleolar, nuclear and cell body size and nuclear position. When isolated, the relatively insoluble cell body cytoskeleton contains Nissl bodies and lipofuscin granules. After axotomy, protein labeling increases markedly and the cytoskeleton enlarges, increasing nucleolar, nuclear and cell body size, as well as nuclear eccentricity. Nearly all of the protein mass that accumulates in the cell body after axotomy appears to be added to the cytoskeleton. Conclusion: We conclude that axotomy causes the conjugate enlargement of the nucleolus, nucleus and cell body and increases nuclear eccentricity in spinal motor neurons by adding protein to the cytoskeleton. The change in nuclear position, we propose, occurs when cytoskeletal elements of the axon cannot enter the shortened axon and "dam up" between the nucleus and axon hillock. As a consequence, we suggest that Nissl body-free axonal cytoskeleton accumulates between the nucleus and axon, displaces Nissl body-containing cytoskeleton, and produces central chromatolysis in that region of the cell. Background known and explored for over a century, these morpholog- Injury to the axon of spinal motor neurons produces ical responses to axotomy have not been adequately major structural changes in the affected cell body. These explained. changes include a transient enlargement of the nucleolus, nucleus and cell body, an increase in nuclear eccentricity It has been proposed that enlargement of the motor neu- and central chromatolysis, which is a centrifugal disap- ron cell body after axotomy is caused by the entry of water pearance of polyribosome-containing Nissl bodies. While into the cell bodies, possibly the result of an early increase Page 1 of 13 (page number not for citation purposes) BMC Neuroscience 2005, 6:19 http://www.biomedcentral.com/1471-2202/6/19 in osmotically-active hydrolytic products of macromole- cules within the motor neurons [1]. On the other hand, the gradual increase in total protein and RNA observed in 6000 axotomized motor neurons cell bodies [2,3] could also 5000 Unextracted play a role in their enlargement after injury. That the Extracted ) nucleolus, nucleus and cell body enlarge together after 3 4000 axotomy suggests that their sizes may be coordinated, 3000 …. especially since a similar scaling is observed in normal, . 2000 non-injured motor neurons of increasing size [4] and in Nucleus (µm motor neurons exposed to excess growth hormone [5]. It 1000 is noteworthy that the nucleus, nucleolus and cell body do 0 not each enlarge after axotomy in all types of neurons 0 10000 20000 30000 40000 capable of axon regeneration, implying that an increase in Cell body volume (µm3) size may not be required for regrowth of the axon [6]. 240 The repositioning of the nucleus in injured motor neu- Unextracted ) 200 rons was quantified by Barr and Hamilton [1], who, like 3 Extracted others [7,8], found that the direction of increased nuclear 160 eccentricity in motor neurons was usually away from the 120 axon hillock. Suggested mechanisms for increased nuclear eccentricity include a selective influx of water into the area 80 of the axon hillock [1] and interference with axonal trans- Nucleolar volume (µm 40 port, leading to the accumulation of axonal constituents 0 in the injured cell body [6]. 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 Nuclear volume (µm3) Central chromatolysis also develops progressively in the region between the nucleus and axon hillock after axotomy [6]. The gradual disappearance of Nissl bodies from PreservationtionshipsFigure 1 in extracted of cell body, spinal nucle motorar and neurons nucleolar volume rela- the perinuclear region towards the periphery of the cell Preservation of cell body, nuclear and nucleolar vol- body and the concomitant loss of basophilic staining of ume relationships in extracted spinal motor neurons. RNA occur even as the radiolabeling and total content of Nuclear volume in individual cell bodies isolated from unfixed RNA increase [6]. Neither the molecular basis of chroma- lumbar spinal cords of normal adult frogs scales with cell tolysis nor the reason for its appearance and centrifugal body volume (top) and nucleolar volume (bottom) before spreading between the nucleus and axon hillock is and after extraction. Significant (p < 0.01) correlation coeffi- known. cients were found in first-order regression analyses for both unextracted (solid line) and extracted cells (dashed line). In this study, we first show that the size and shape of normal, non-injured frog motor neuron cell bodies are maintained by a cytoskeleton that can be isolated. We then provide evidence that axotomy increases the synthesis and total content of proteins in the cytoskeleton, which range of cell body areas in motor neurons isolated from appears to alter its structure and produce nucleolar, normal animals by the method of Sinicropi and McIlwain nuclear and cell body enlargement. We further show that [10] is slightly larger than that of their fixed, sectioned the cytoskeleton maintains nuclear position in the unin- counterparts [5]. As with area measurements on normal jured cell body and is altered by axotomy to increase motor neurons [5], nucleolar and nuclear volume increase nuclear eccentricity. Finally, we propose that both nuclear as cell body volume increases (Fig. 1). Since the plasma eccentricity and central chromatolysis result from an accu- membrane is damaged during the isolation of motor neu- mulation of axonal cytoskeleton that is impeded from ron cell bodies and is no longer semipermeable to most entering the truncated axon. osmolytes [11], osmotic forces do not appear to be chiefly responsible for maintaining cell body size in isolated Results motor neurons. Even when intracellular membranes are The cytoskeleton maintains nucleolar, nuclear and cell permeabilized with 1% Triton X-100 and over one-half of body size in normal motor neurons the cell body protein is removed from isolated motor neu- Isolated, unfixed motor neurons, like fixed, sectioned rons by successive extraction and high salt solutions, the motor neurons, vary widely in cell body area [5,9]. The mean size of the nucleolus, nucleus and cell body is little Page 2 of 13 (page number not for citation purposes) BMC Neuroscience 2005, 6:19 http://www.biomedcentral.com/1471-2202/6/19 Table 1: The cytoskeleton maintains most of cell body, nuclear and nucleolar volume in normal motor neurons. Unextracted Volume (µm3) Extracted Volume (µm3)E/Ua Nucleolus 74.5 ± 11.0 86.8 ± 18.3 1.17 Nucleus 2,402 ± 423 1,968 ± 339** 0.82 Cell Body 19,387 ± 2,160 16,409 ± 1,662** 0.85 Mean volume ± S.D from 6 experiments; approximately 50 lumbar cell bodies from 3 frogs were analyzed in each experiment. Isolated cell bodies were extracted by the method of He et al. [12] to obtain cytoskeletons. a E/U = extracted/unextracted **p < 0.01, Student's paired t-test, extracted vs. unextracted counterpart affected (Table 1). This extraction procedure, which has been used to isolate the nuclear matrix and cytoplasmic skeleton of non-neuronal cells [12], reduces the cell body protein content in normal frog motor neurons from 4.35 ± 0.87 to 1.88 ± 0.29 ng/cell (a 56.8% decrease). In contrast, cell body and nuclear volume decline only by 15 and 17%, respectively, and nucleolar volume does not change significantly (Table 1). The volumes of the nucleolus and nucleus in the extracted motor neurons continue to scale with cell body volume (Fig. 1) and the extracted cells have a relatively normal appearance by differential interference contrast microscopy (Fig. 2). These findings suggest that nucleolar, nuclear and cell body size in motor neurons are maintained by cytoskeletal structures and led us to exam- ine the ultrastructure of extracted motor neurons. Resinless thin sections [12] were prepared from frog lumbar spinal cord that had been

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